Fucoidan alleviated autoimmune diabetes in NOD mice by regulating pancreatic autophagy through the AMPK/mTOR1/TFEB pathway.

Objectives: The present study investigated the effect and its underlying mechanisms of fucoidan on Type 1 diabetes mellitus (T1DM) in non-obese diabetic (NOD) mice. Materials and Methods: Twenty 7-week-old NOD mice were used in this study, and randomly divided into two groups (10 mice in each group): the control group and the fucoidan treatment group (600 mg/kg. body weight). The weight gain, glucose tolerance, and fasting blood glucose level in NOD mice were detected to assess the development of diabetes. The intervention lasted for 5 weeks. The proportions of Th1/Th2 cells from spleen tissues were tested to determine the anti-inflammatory effect of fucoidan. Western blot was performed to investigate the expression levels of apoptotic markers and autophagic markers. Apoptotic cell staining was visualized through TdT-mediated dUTP nick-end labeling (TUNEL). Results: The results suggested that fucoidan ameliorated T1DM, as evidenced by increased body weight and improved glycemic control of NOD mice. Fucoidan down-regulated the Th1/Th2 cells ratio and decreased Th1 type pro-inflammatory cytokines' level. Fucoidan enhanced the mitochondrial autophagy level of pancreatic cells and increased the expressions of Beclin-1 and LC3B II/LC3B I. The expression of p-AMPK was up-regulated and p-mTOR1 was inhibited, which promoted the nucleation of transcription factor EB (TFEB), leading to autophagy. Moreover, fucoidan induced apoptosis of pancreatic tissue cells. The levels of cleaved caspase-9, cleaved caspase-3, and Bax were up-regulated after fucoidan treatment. Conclusion: Fucoidan could maintain pancreatic homeostasis and restore immune disorder through enhancing autophagy via the AMPK/mTOR1/TFEB pathway in pancreatic cells.

TLR5-deficiency controls dendritic cell subset development in an autoimmune diabetes-susceptible model.

Introduction: The incidence of the autoimmune disease, type 1 diabetes (T1D), has been increasing worldwide and recent studies have shown that the gut microbiota are associated with modulating susceptibility to T1D. Toll-like receptor 5 (TLR5) recognizes bacterial flagellin and is widely expressed on many cells, including dendritic cells (DCs), which are potent antigen-presenting cells (APCs). TLR5 modulates susceptibility to obesity and alters metabolism through gut microbiota; however, little is known about the role TLR5 plays in autoimmunity, especially in T1D. Methods: To fill this knowledge gap, we generated a TLR5-deficient non-obese diabetic (NOD) mouse, an animal model of human T1D, for study. Results: We found that TLR5-deficiency led to a reduction in CD11c+ DC development in utero, prior to microbial colonization, which was maintained into adulthood. This was associated with a bias in the DC populations expressing CD103, with or without CD8α co-expression, and hyper-secretion of different cytokines, both in vitro (after stimulation) and directly ex vivo. We also found that TLR5-deficient DCs were able to promote polyclonal and islet antigen-specific CD4+ T cell proliferation and proinflammatory cytokine secretion. Interestingly, only older TLR5-deficient NOD mice had a greater risk of developing spontaneous T1D compared to wild-type mice. Discussion: In summary, our data show that TLR5 modulates DC development and enhances cytokine secretion and diabetogenic CD4+ T cell responses. Further investigation into the role of TLR5 in DC development and autoimmune diabetes may give additional insights into the pathogenesis of Type 1 diabetes.

Refined protocol for newly onset identification in non-obese diabetic mice: an animal-friendly, cost-effective, and efficient alternative.

BACKGROUND: Therapeutic interventions for diabetes are most effective when administered in the newly onset phase, yet determining the exact onset moment can be elusive in practice. Spontaneous autoimmune diabetes among NOD mice appears randomly between 12 and 32 weeks of age with an incidence range from 60 to 90%. Furthermore, the disease often progresses rapidly to severe diabetes within days, resulting in a very short window of newly onset phase, that poses significant challenge in early diagnosis. Conventionally, extensive blood glucose (BG) testing is typically required on large cohorts throughout several months to conduct prospective survey. We incorporated ultrasensitive urine glucose (UG) testing into an ordinary BG survey process, initially aiming to elucidate the lag period required for excessive glucose leaking from blood to urine during diabetes progression in the mouse model. RESULTS: The observations unexpectedly revealed that small amounts of glucose detected in the urine often coincide with, sometimes even a couple days prior than elevated BG is diagnosed. Accordingly, we conducted the UG-based survey protocol in another cohort that was validated to accurately identified every individual near onset, who could then be confirmed by following few BG tests to fulfill the consecutive BG + criteria. This approach required fewer than 95 BG tests, compared to over 700 tests with traditional BG survey, to diagnose all the 37-38 diabetic mice out of total 60. The average BG level at diagnosis was slightly below 350 mg/dl, lower than the approximately 400 mg/dl observed with conventional BG monitoring. CONCLUSIONS: We demonstrated a near perfect correlation between BG + and ultrasensitive UG + results in prospective survey with no lag period detected under twice weekly of testing frequency. This led to the refined protocol based on surveying with noninvasive UG testing, allowing for the early identification of newly onset diabetic mice with only a few BG tests required per mouse. This protocol significantly reduces the need for extensive blood sampling, lancet usage, labor, and animal distress, aligning with the 3Rs principle. It presents a convenient, accurate, and animal-friendly alternative for early diabetes diagnosis, facilitating research on diagnosis, pathogenesis, prevention, and treatment.

Profiling the Gut Microbiome Unraveled Signature Bacterial Groups in Autoimmune Diabetes, which Remain Unperturbed by the Low Ionic Strength of the Drinking Water in NOD mice.

BACKGROUND: Non-obese diabetic (NOD) mice develop type 1 diabetes (T1D) spontaneously and serve as a good model for investigating the underlying pathological mechanisms and devising novel treatment procedures. Although acid water consumption has been reported to exaggerate or reduce diabetes incidence in female NOD mice by two groups, the causative bacteria responsible for these contrasting changes remain unclear. On the contrary, we and others failed to observe the effect of acid water consumption on diabetes incidence. This study aimed to determine whether the consumption of low-pH drinking water could alter the frequencies of prominent bacterial groups independent of diabetes manifestation. METHODS: Six-week-old female NOD mice maintained on acidified drinking water at the Jackson Laboratories were transferred to neutral pH water or continuously provided with low pH drinking water at our facility. Diabetes was monitored weekly using a glucometer. Using the 454-pyrosequencing methodology, we profiled the gut microbiome of mice transferred to neutral water and developed diabetes. Further, we performed quantitative real-time polymerase chain reactions (qRT-PCR) using primers specific for prominent 16S rRNA genes on the fecal DNA of mice provided with low pH or neutral water and displayed diabetes similarly. RESULTS: Consistent with our earlier report, the incidence of T1D was robust (80-100%) regardless of whether female NOD mice consumed acid (~pH 2.9) or neutral water. The 454-pyrosequencing of fecal DNA indicated no substantial influence of transferring mice to neutral pH drinking water on the gut microbiome. To validate these findings, we conducted qRT-PCR on the fecal DNA of mice longitudinally from six weeks of age to adulthood that consumed acidic or neutral pH water and developed diabetes similarly. Among the 15 selected bacterial groups examined, the frequency of Lactobacillus sp. remained consistently lower (p < 0.05) throughout the life of NOD mice compared to that found in young (6-week-old) mice, regardless of the pH of the drinking water. The relative frequencies of the Firmicutes Ruminococcaceae and the Bactereoidetes members Anaerophaga sp. and Paludibacter sp. increased significantly (p < 0.05) during the transition to the overtly diabetic stage irrespective of the ionic strength of the drinking water. Interestingly, the Firmicutes members Clostridium coccoides, C. leptum, and Lachnospiraceae and the Bacteroidetes members Bacteroides sp. and Prevottella sp. remained unchanged throughout the analysis irrespective of the pH of the drinking water. Paradoxically, the representations of Akkermansia muciniphila and the segmented filamentous bacteria implicated in diabetes protection did not differ regardless of the age or the ionic strength of the drinking water. CONCLUSIONS: The data presented herein validate the lack of influence of acidic drinking water on T1D development in female NOD mice. Diabetes was associated with the lower representation of Lactobacillus sp. throughout life, which was not influenced by the differing pH of the drinking water. Significantly, segmented filamentous bacteria and A. muciniphila, previously implicated in protection against T1D, were not perturbed by the varying pH of the water consumed. These data indicate that although acidified water consumption was reported previously to diminish specific gastrointestinal pathogens, it failed to perturb gut commensals that influence diabetes development.

Gut microbiota from B-cell-specific TLR9-deficient NOD mice promote IL-10+ Breg cells and protect against T1D.

Introduction: Type 1 diabetes (T1D) is an autoimmune disease characterized by the destruction of insulin-producing ß cells. Toll-like receptor 9 (TLR9) plays a role in autoimmune diseases, and B cell-specific TLR9 deficiency delays T1D development. Gut microbiota are implicated in T1D, although the relationship is complex. However, the impact of B cell-specific deficiency of TLR9 on intestinal microbiota and the impact of altered intestinal microbiota on the development of T1D are unclear. Objectives: This study investigated how gut microbiota and the intestinal barrier contribute to T1D development in B cell-specific TLR9-deficient NOD mice. Additionally, this study explored the role of microbiota in immune regulation and T1D onset. Methods: The study assessed gut permeability, gene expression related to gut barrier integrity, and gut microbiota composition. Antibiotics depleted gut microbiota, and fecal samples were transferred to germ-free mice. The study also examined IL-10 production, Breg cell differentiation, and their impact on T1D development. Results: B cell-specific TLR9-deficient NOD mice exhibited increased gut permeability and downregulated gut barrier-related gene expression. Antibiotics restored gut permeability, suggesting microbiota influence. Altered microbiota were enriched in Lachnospiraceae, known for mucin degradation. Transferring this microbiota to germ-free mice increased gut permeability and promoted IL-10-expressing Breg cells. Rag-/- mice transplanted with fecal samples from Tlr9 fl/fl Cd19-Cre+ mice showed delayed diabetes onset, indicating microbiota's impact. Conclusion: B cell-specific TLR9 deficiency alters gut microbiota, increasing gut permeability and promoting IL-10-expressing Breg cells, which delay T1D. This study uncovers a link between TLR9, gut microbiota, and immune regulation in T1D, with implications for microbiota-targeted T1D therapies.

Tityus serrulatus envenoming in non-obese diabetic mice: a risk factor for severity

Background In Brazil, accidents with venomous animals are considered a public health problem. Tityus serrulatus (Ts), popularly known as the yellow scorpion, is most frequently responsible for the severe accidents in the country. Ts envenoming can cause several signs and symptoms classified according to their clinical manifestations as mild, moderate or severe. Furthermore, the victims usually present biochemical alterations, including hyperglycemia. Nevertheless, Ts envenoming and its induced hyperglycemia were never studied or documented in a patient with diabetes mellitus (DM). Therefore, this is the first study to evaluate the glycemia during Ts envenoming using a diabetic animal model (NOD, non-obese diabetic). Methods Female mice (BALB/c or NOD) were challenged with a non-lethal dose of Ts venom. Blood glucose level was measured (tail blood using a glucose meter) over a 24-h period. The total glycosylated hemoglobin (HbA1c) levels were measured 30 days after Ts venom injection. Moreover, the insulin levels were analyzed at the glycemia peak. Results The results demonstrated that the envenomed NOD animals presented a significant increase of glycemia, glycosylated hemoglobin (HbA1c) and insulin levels compared to the envenomed BALB/c control group, corroborating that DM victims present great risk of developing severe envenoming. Moreover, the envenomed NOD animals presented highest risk of death and sequelae. Conclusions This study demonstrated that the diabetic victims stung by Ts scorpion should be always considered a risk group for scorpion envenoming severity.(AU)

Tityus serrulatus envenoming in non-obese diabetic mice: a risk factor for severity

In Brazil, accidents with venomous animals are considered a public health problem. Tityus serrulatus (Ts), popularly known as the yellow scorpion, is most frequently responsible for the severe accidents in the country. Ts envenoming can cause several signs and symptoms classified according to their clinical manifestations as mild, moderate or severe. Furthermore, the victims usually present biochemical alterations, including hyperglycemia. Nevertheless, Ts envenoming and its induced hyperglycemia were never studied or documented in a patient with diabetes mellitus (DM). Therefore, this is the first study to evaluate the glycemia during Ts envenoming using a diabetic animal model (NOD, non-obese diabetic). Methods Female mice (BALB/c or NOD) were challenged with a non-lethal dose of Ts venom. Blood glucose level was measured (tail blood using a glucose meter) over a 24-h period. The total glycosylated hemoglobin (HbA1c) levels were measured 30 days after Ts venom injection. Moreover, the insulin levels were analyzed at the glycemia peak. Results The results demonstrated that the envenomed NOD animals presented a significant increase of glycemia, glycosylated hemoglobin (HbA1c) and insulin levels compared to the envenomed BALB/c control group, corroborating that DM victims present great risk of developing severe envenoming. Moreover, the envenomed NOD animals presented highest risk of death and sequelae. Conclusions This study demonstrated that the diabetic victims stung by Ts scorpion should be always considered a risk group for scorpion envenoming severity.

Comparação dos efeitos do gangliosideo GM1 e do fator de crescimento neural (NGF) sobre a expressão de receptor de alta afinidade para NGF, TrkA e insulina em ilhotas pancreaticas isoladas de camundongos NOD (diabetico não obeso) / Comparison of the effect of ganglioside GM1 and the Nerve Growth Factor (NGF) on the expression of receiver of high affinity for NGF, TrkA and insulin in isolated pancreatic islets of NOD mice (non obese diabetic)

O camundongo não obeso diabético (NOD) é caracterizado por desenvolver naturalmente diabetes mellitus tipo 1 (DM-1) com similaridade ao diabetes mellitus tipo 1 em humanos. A manifestação espontânea do diabetes neste modelo animal é caracterizado por infiltração progressiva das ilhotas de Langerhans por células mononucleares linfócitos T (CD4+ e CD8+) e destruição das células ß pancreáticas produtoras de insulina. O fator de crescimento neural (NGF) e algumas citocinas estão associados a regeneração neural, além de atuarem sobre células do sistema imune. Em adição a estes efeitos, NGF age na liberação de insulina pelas células betas das ilhotas pancreáticas, tornando-se foco de interesse com relação as suas propriedades moduladoras no processo inflamatório na ilhota pancreática. O gangliosídeo GM1 liga-se ao receptor de alta afinidade (TrkA) do NGF-ß, mimetizando seus efeitos. No presente trabalho, avaliamos a ação modulatória de GM1 e NGF em cultura de ilhotas pancreáticas, provenientes de camundongos NOD. Foram avaliados por meio de RT-PCR a expressão gênica de NGF-ß, TrkA e insulina e, por ensaio imunoenzimático, a concentração de citocinas IL-1ß, IL-12, TNF-a, INF-y e insulina. Nossos resultados sugerem ação moduladora similar entre GM1 e NGF sobre as ilhotas de NOD não diabéticos e pré-diabéticos. NGF e GM1 aumentam a expressão gênica de NGF e TrkA e diminuem a expressão gênica de insulina em NOD não diabéticos e pré-diabéticos. Além disso, aumentam a liberação de insulina e diminui a de citocinas inflamatórias IL-1ß, IL-12, TNF-a, IFN-y que caracterizam a resposta Th1.

The non-obese diabetic mice (NOD) lineage is characterized by developing type 1 diabetes mellitus (DM-1) naturally, bearing a similarity to DM-1 in human beings. The spontaneous manifestation of diabetes is characterized by gradual infiltration in pancreatic islets by mononuclear cells lymphocytes T (CD4+ and CD8+) and destruction of the ß-cells producers of insulin. One consequence of this effect, is the release of neurotrophins trying modulate the insulin release by the ß cells of pancreatic islets. Thus, the neurotrophins have been the focus of interest in the modulation of the inflammatory process in the pancreatic islets. The ganglioside GM1 binds to the high affinity receptor (TrkA) of the NGF-ß, enhancing its effect. In the present work, we evaluate the immune modulation properties of GM1 and NGF in culture of pancreatic islets from NOD mice. The gene expression of NGF-ß, TrkA and insulin for immune enzymatic assay, the concentration of cytokines IL 1ß, IL-12, TNF-a, IFN-y and insulin were evaluated by RT-PCR and ELISA. Our results suggest similar modulation action between GM1 and NGF on islets of NOD non-diabetic and pre-diabetic. GM1 and NGF action increases the gene expression of NGF and TrkA and the decrease of insulin in mice NOD non-diabetic and pre-diabetic. Moreover, GM1 and NGF increase the insulin release and decrease inflammatory cytokines that characterize the Th1 reply.